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Control of Eukaryotic Translation SHOWCASE ON RESEARCH Control of Eukaryotic Translation Thomas Preiss1,2 1Molecular Genetics Program, Victor Chang Cardiac Research Institute, NSW 2010 2School of Biotechnology & Biomolecular Sciences and St Vincent's Clinical School, University of New South Wales, NSW 2052 A common view holds that most control mechanisms This is particularly true during translation initiation on to regulate eukaryotic gene expression target the eukaryotic mRNAs (Fig. 2). This process depends on primary step, namely transcription in the nucleus. In the 5' m7G(5')ppp(5')N cap structure and the 3' poly(A) contrast to this, it is becoming increasingly apparent tail of a typical mRNA and at least 12 eukaryotic that controls acting on post-transcriptional steps of initiation factors (eIFs) (1, 2). Initiation begins with the mRNA metabolism, in particular at the level of binding of several eIFs and other components to the translation, are also of critical importance (Fig. 1). small (40S) ribosomal subunit. This complex is Translation is carried out on the ribosomes and is recruited to the (capped) 5' end of the mRNA, then usually divided into three phases: (i) initiation, (ii) 'scans' the 5' untranslated region (UTR) of the mRNA elongation and (iii) termination. The initiation phase and recognises the start codon. Joining of a large (60S) represents all processes required for the assembly of a subunit completes the assembly of a complete (80S) ribosome at the start codon of the mRNA. The actual ribosome. The 40S subunit is primed for initiation polypeptide synthesis takes place during the elongation through binding of a ternary complex comprising eIF2, Met phase. When ribosomes reach the stop codon this Met-tRNAi and GTP. The mRNA is prepared by the signals termination − the dissociation of the completed action of the eIF4 group of factors. eIF4E binds the cap polypeptide and the ribosome from the mRNA. Why structure and eIF4A is an ATP-dependent RNA control translation? The best answer to this question helicase that is able, upon stimulation by eIF4B, to probably is that it affords desirable complexity to gene unwind secondary structure in the cap-proximal regulation. There are several features of translational region of the mRNA (Fig. 2A). eIF4G is an adaptor control that are particularly advantageous in certain protein that interacts with eIF4E, eIF4A, and eIF3, cellular situations. It is a fast response, which may another 40S-bound factor. The poly(A) tail-binding explain why it is commonly involved in cellular stress protein PABP also has a critical function at this stage: it responses. Translation can also be controlled locally in helps to recruit eIF4G and confers a pseudo-circular areas distant from the nucleus, for instance to support conformation to the mRNA, the exact functional synaptic function in the nervous system. Furthermore, it significance of which still remains to be determined. can operate in the absence of nuclear activity, a feature Scanning of the mRNA 5' UTR by the 40S subunit o f e a r l y d e v e l o p m e n t o r t h e l a t e s t a g e s o f requires contributions by several of the assembled eIFs erythropoiesis. (Fig. 2B). Base-pairing between the start codon and Met anticodon loop of the Met-tRNAi triggers GTP The Mechanism of Translation Initiation hydrolysis by eIF2, dissociation of eIFs and 60S subunit Ribosomes cannot carry out their functions alone; they joining (Fig. 2C). A second GTP hydrolysis step by depend on auxiliary factors that help them to engage the eIF5B completes 80S ribosome assembly. An important mRNA template, to select the activated building blocks feature of eIF2 is that it requires eIF2B to exchange for polypeptide synthesis and to mediate termination. bound GDP for GTP after each round of initiation. Fig. 1. 'Strict Tempo' models of eukaryotic gene regulation. Contrary to an extreme view (step diagram on the left), the o u t c o m e o f t h e g e n e expression cascade does not only depend on mechanisms to control gene transcription. Instead, virtually all aspects of m R N A a n d p r o t e i n metabolism are subject to controlling influences that can affect the outcome of gene expression in a quantitative and qualitative manner (step diagram on the right). Vol 36 No 3 December 2005 AUSTRALIAN BIOCHEMIST Page 9 SHOWCASE ON Control of Eukaryotic Translation RESEARCH altering availability or function of eIFs, most commonly eIF2 and eIF4E. mRNA-specific control typically involves regulatory complexes that recognize particular elements, usually in the 5' or 3' UTR of the mRNA, and exclusively alter translation of the targeted mRNAs. Regulatory elements found in the 5' UTR of mRNAs include repressor protein binding sites, inhibitory RNA structural features, upstream AUG (uAUG) or upstream short open reading frames (uORF) and internal ribosome entry sites (IRES). Bound repressors and RNA structures serve as steric 'roadblocks' that hinder the normal progression of initiation, while uAUGs and uORFs typically engage a proportion of scanning ribosomes in non-productive initiation events that lower translation of the main ORF. IRES elements are complex RNA structures that can recruit the translation initiation machinery directly to internal positions on the mRNA, bypassing the need for the cap structure at the 5' end of the mRNA. 3' UTR elements often recruit regulatory complexes that affect translation by forming a bridging interaction with initiation intermediates at the 5' end. Examples of translational control that illustrate aspects of these generic descriptions are presented below. Global Control A common means to achieve global control of translation is through changes in the phosphorylation state of eIFs or the regulators that act on them. Mammalian cells contain several kinases that phosphorylate the α subunit of eIF2, leading to a block of the GDP/GTP exchange reaction and inhibition of global translation. Each kinase is activated in response to specific cellular stress conditions: PKR (protein kinase activated by double-stranded RNA) is activated during viral replication; GCN2 (general control non- derepressible 2) is stimulated by amino acid starvation; PERK (PKR-like endoplasmic reticulum eIF2α kinase) senses unfolded protein accumulation in the endoplasmic reticulum; HRI (heme-regulated inhibitor) Fig. 2. The initiation phase of translation. reacts to heme depletion. Appropriate control of eIF2 (A) An early step in initiation is the binding of the eIF4 and eIF2B is important for normal physiology and factors to the cap structure followed by unwinding mutations in the genes for PERK or eIF2B give rise to of secondary structure in the mRNA 5' UTR. The serious human disease (4). interaction of PABP with eIF4G aids this process Extracellular cues such as insulin and growth factors and leads to circularisation of the mRNA. activate the PI3K/AKT/mTOR and Ras/MAPK (B) The 40S ribosomal subunit, associated eIFs, Met- signaling pathways that also modulate translation (5, Met 6 ) . R a s / M A P K s i g n a l l i n g i n c r e a s e s e I F 4 E tRNAi , and GTP are recruited and this complex moves along the mRNA in a 3' direction. phosphorylation (and thus translation) through the (C) Identification of the AUG start codon leads to the eIF4G-bound Mnk1/2 kinases. The mTOR pathway release of eIFs, GTP hydrolysis and binding of the leads to phosphorylation of the 4E-binding proteins, a 60S ribosomal subunit. The simplified diagrams group of small regulatory proteins that mimic the part only show a selection of participating eIFs. o f e I F 4 G t h a t i n t e r a c t s w i t h e I F 4 E ( F i g . 3 A ) . Hypophosphorylated 4E-BPs bind to eIF4E and Strategies for Translational Control competitively displace eIF4G, resulting in inhibition of translation. Hyperphosphorylated 4E-BPs are released Initiation is usually the rate-limiting step of from eIF4E, leading to activation of translation (7). translation and the most common target of regulatory Translational control through these pathways is critical intervention. Translational control mechanisms may be for appropriate regulation of cell growth. Its broadly divided into global and mRNA-specific types deregulation is involved in cancer biology (8) and of control (3). Global control affects the translation of pathological cardiac hypertrophy (9, 10). most cellular mRNAs and is usually achieved by Page 10 AUSTRALIAN BIOCHEMIST Vol 36 No 3 December 2005 SHOWCASE ON Control of Eukaryotic Translation RESEARCH Proteolytic cleavage of eIFs is also used to alter cellular translation (11). During apoptosis, caspase-3 cleaves both mammalian isoforms of eIF4G at multiple positions. Caspase-3 also cleaves the eIF4G- homologous protein, death associated protein-5 (DAP5) near its C-terminus, which may activate its function as a specialised initiation factor. Although these global controls all affect general translation, there is mounting evidence that they affect the translation of specific mRNAs in a particular manner. Such mRNAs may have structural features and/or regulatory elements that make their translation particularly sensitive or resistant to a global change in cellular translation. mRNA-Specific Control The paradigm of mRNA-specific control in the context of general eIF regulation is GCN4 mRNA, which encodes a transcriptional activator of amino acid biosynthesis genes in yeast (3, 12). The 5' region of GCN4 mRNA contains four uORFs, which collectively lead to low levels of Gcn4p synthesis u n d e r n o r m a l c o n d i t i o n s a n d , s o m e w h a t paradoxically, increased levels during amino acid starvation.
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